FIELD STUDIES AND MONITORING OF MOSQUITO POPULATIONS (DIPTERA: CULICIDAE) IN URBAN ENVIRONMENTS Except where reference is made to the work of others, the work described in this thesis is my own or was done in collaboration with my advisory committee. This thesis does not include proprietary or classified information. _______________________________________ Whitney Allyn Qualls Certificate of Approval: ___________________________ ___________________________ George W. Folkerts Gary R. Mullen, Chair Profesor Profesor Biology Entomology and Plant Pathology ____________________________ ___________________________ Wayne E. Clark Stephen L. McFarland Profesor Acting Dean Entomology and Plant Pathology Graduate School FIELD STUDIES AND MONITORING OF MOSQUITO POPULATIONS (DIPTERA: CULICIDAE) IN URBAN ENVIRONMENTS Whitney Allyn Qualls A Thesis Submitted to the Graduate Faculty of Auburn University in Partial Fulfillment of the Degree of Master of Science Auburn, Alabama December 16, 2005 iii FIELD STUDIES AND MONITORING OF MOSQUITO POPULATIONS (DIPTERA: CULICIDAE) IN URBAN ENVIRONMENTS Whitney Allyn Qualls Permission is granted to Auburn University to make copies of this thesis at its discretion, upon request of individuals or institutions and at their expense. The author reserves all publication rights. ______________________________ Signature of Author ______________________________ Date VITA iv Whitney Allyn Qualls, daughter of Jerry Allen Qualls and Sara Jo (Costner) Qualls, was born December 31, 1980, in Athens, Tennessee. She graduated from McMinn Central High school in 1999. She attended Cumberland College in Williamsburg, Kentucky, graduating in 2003 with a Bachelor of Science Degree in Biology. In 2003 she entered the Master of Science program in the Department of Entomology and Plant Pathology at Auburn University. v THESIS ABSTRACT FIELD STUDIES AND MONITORING OF MOSQUITO POPULATIONS (DIPTERA: CULICIDAE) IN URBAN ENVIRONMENTS Whitney Allyn Qualls Master of Science, December 16, 2005 (B.S., Cumberland College, 2003) 97 Typed Pages Directed by Gary R. Mullen In 2004 and 2005 field work was conducted to survey and monitor larval and adult mosquito populations in urban habitats with, primary interest in Aedes albopictus, the Asian tiger mosquito. In 2004 and 2005 a state-wide survey of tire-breeding mosquitoes was conducted. Tire sites in all 67 counties in the state of Alabama were sampled for mosquito larvae. A total of 13,022 mosquito larvae, representing 13 mosquito species in 7 genera, was collected. The most frequently collected species were Ae. albopictus (71%),Culex territans (7.5%), and Ochlerotatus triseriatus (7.1%). The following species were also collected: Cx. restuans (5.2%), Cx. salinarius (3.5%), Orthopodomyia signifera (2.7%), Cx. quinquefasciatus (1.2%), Oc. atropalpus (<1%), Toxorhynchites rutilus (<1%) vi Anopheles punctipennis (<1%), An. quadrimaculatus (<1%), An. spp. (<1%), and Psorophora columbiae (<1%). No Ae. aegypti or Oc. japonicus were collected from tires during this survey. Psorophora columbiae was also collected from discarded tires, representing the first recorded collection of this species from tires. This study demonstrated that since the first detection of Ae. albopictus at Cullman, Alabama in 1985, Ae. albopictus has established itself throughout the state becoming the most common tire-inhabiting mosquito in Alabama. Results also showed that the yellow fever mosquito Ae. aegypti is no longer the dominant tire-breeder in Alabama. In fact it appears that Ae. aegypti has been displaced from tires throughout state. In 2004, studies were conducted to evaluate the short-range mosquito attractant 1- octeno-3-ol (octenol) used with commercially available propane-powered mosquito traps to increase collections of urban mosquitoes. Octenol was evaluated using the Mosquito Magnet Pro TM (MMP) trap. Three field trials were conducted in the communities of Auburn and Phenix City, Alabama. Four MMP traps were placed in a 1x2 factorial design. Aedes albopictus, Coquillettida perturbans, and Oc. triseriatus collections were significantly enhanced with octenol as determined with a 3-way ANOVA, P < .05. Anopheles punctipennis, Ps. columbiae, Cx. restuans, and Cx. salinarius collections were also significantly enhanced with octenol as determined by a Chi-square analysis, P < .05. Twelve out of 13 mosquito species were collected in greater numbers with octenol than without octenol throughout this study. vii ACKNOWLEDGEMENTS The author would like to thank her advisor Gary R. Mullen, her committee members George W. Folkerts and Wayne E. Clark; Ashley Lovell and the Alabama Department of Public Health for assistance in organizing the state-wide tire-breeding mosquito survey; and Matthew Smith, Scott Croxton, Will Sherrer and Nathan Burkett who helped in field work. She also would like to thank her parents for their love and support. viii Style manual or journal used: Journal of the American Mosquito Control Association Computer software used: Microsoft Word ? ix TABLE OF CONTENTS LIST OF TABLES??????????????????????????......xi LIST OF FIGURES??????????.?????????????.....?....xiv I. INTRODUCTION??????????????????????.???..1 Literature Cited????????????????????????.?4 II. STATE-WIDE SURVEY OF TIRE-BREEDING MOSQUITOES (DIPTERA: CULICIDAE) IN ALABAMA??????.??????..???.7 Materials and Methods???.??????????????.???.....11 Results ??????????.???????????.........???.....12 Discussion???????..???????????????.??.......14 Literature Cited???????????????????...????...19 III. EVALUATION OF THE MOSQUITO ATTRACTANT OCTENOL FOR COLLECTING URBAN MOSQUITOES???????...54 Study Sites?????????????????????..??...?....57 Materials and Methods???????????????..???..?......59 Results ???????????..???????????..????..61 Discussion????????????????..????..???.........64 x Literature Cited????????????????????????.67 APPENDX?????????????????????????????...81 xi LIST OF TABLES 1. Total number and percentage of mosquito species collected from tires in Alabama by month, based on the 2004-2005 larval survey. Species are listed in descending order of the total numbers of each species collected................................................23 2. Total number of mosquitoes species collected by county and month from tires in Alabama, based on the 2004-2005 larval survey. The sixty-seven Alabama counties are listed in alphabetical order ..????????????...??.....24 3. Presence or absence of tire-breeding mosquitoes by 2-week intervals from May through October, based on the 2004-2005 larval survey. Species are listed in descending order of the total numbers of each species collected??????.....40 4. Total number of mosquito species collected by physiogeographic region and month from tires in Alabama, based on the 2004-2005 larval survey.????....41 5. Presence or absence of tire-breeding mosquitoes collected in each physiographic region by 2-week intervals from May through October, based on the 2004-2005 larval survey ???????????.?...........................................................42 xii 6. Mosquito species collected with and without octenol at Field Site 1, June 2004. Species are listed in descending order of the total number of each species collected?????????????????...?????.??..???.69 7. Repeated measures ANOVA values for mosquito species with trap collections >100 specimens at Field Site 1???????...???????????..?70 8. Mosquito species collected with and without octenol at Field Site 2, June-July 2004. Species are listed in descending order of the total number of each species collected.?...???????????????.?....71 9. Repeated measures ANOVA values for mosquito species with trap collections >100 specimens at Field Site 2.???..????...?..??...................?..........72 10. Mosquito species collected with and without octenol at Field Site 3, July-August, 2004. Species are listed in descending order of the total number of each species collected??????????????????..???....73 11. ANOVA values for mosquito species with trap collections > 100 specimens at Field Site 3???................................................................??.............??.....74 xiii 12. Mosquito larval samples collected from tires at the parts-and-salvage yard in Phenix City, Alabama, during field trials evaluating the Mosquito Magnet Pro TM traps with and without octenol.?...........................................................................75 xiv LIST OF FIGURES 1. Map of Alabama counties reproduced from Department of Geography, College of Arts and Sciences, the University of Alabama?.....................................44 2. Map of Alabama physiographic regions reproduced from the Department of Geography, College of Arts and Sciences, the University of Alabama????...45 3. Alabama counties in which larvae were collected from tires, shown in gray. a, 2004. b,2005.????..??????????...??.???.????.......46 4. Map of Alabama showing number of tire sites sampled per county during the 2004-2005 larval survey. A total of 169 tire sites sampled???.????...47 5. Distribution, by county, of tire-breeding mosquitoes in Alabama, based on 2004- 2005 larval survey. Aedes albopictus, Cx. quinquefasciatus, Cx. restuans, and Cx. salinarius.??????..?....................................................................................48 6. Distribution, by county, of tire-breeding mosquitoes in Alabama, based on 2004- 2005 larval survey. Culex territans, Oc. atropalpus, Oc. triseriatus, and Or. signifera .??????...????????????????????...?.49 xv 7. Distribution, by county, of tire-breeding mosquitoes in Alabama, based on 2004- 2005 larval survey. Psorophora columbiae, Tx. rutilus, An. punctipennis, and An. quadrimaculatus???.???. ???..????..????????50 8. Distribution, by county, of tire-breeding mosquitoes in Alabama, based on 2004- 2005 larval survey. Anopheles spp?.????????????????..?.51 9. Occurrence of tire-breeding mosquitoes by 2-week intervals from May through October, based on the 2004-2005 larval survey. Species are listed in descending ???????...???????????..???.?.???......52 10. Map of Alabama showing the 5 physiographic regions and the number of tire sites sampled per county by physiographic region based on the 2004-2005 larval survey???????...????????????????..????......53 11. American Biophysics Corporation?s propane-powered Mosquito Magnet Pro TM trap???........?????????????????????????....76 12. Collections of landing mosquitoes using a hand-held, battery-operated aspirator to remove landing females during a 2-minute period to determine biting activity at a parts-and-salvage yard, in Phenix City, Alabama, 2004?????????.?77 xvi 13. Collections of Aedes albopictus with and without octenol at Field Site 1 during successive trap nights. Collections of Aedes albopictus (black) were made with octenol. Collections of Ae. albopictus (gray) were made without octenol. Five trap- night collections were significantly enhanced with octenol (P < .05), as indicated by asterisks, based on a Chi-square analysis.????.........????.....??...78 14. Collections of Aedes albopictus with and without octenol at Field Site 2 during successive trap nights. Collections of Aedes albopictus (black) were made with octenol. Collections of Ae. albopictus (gray) were made without octenol. Nine trap-night collections were significantly enhanced with octenol (P < .05), as indicated by asterisks, based on a Chi-square analysis...??????........?...79 15. MMP collections compared to mean landing counts of Ae. albopictus by trap week. MMP collections on the left y-axis and mean landing-count collections on right y-axis of Aedes albopictus at Field Site 3. Based on a Proc Corr analysis, the mean landing counts of Ae. albopictus were significantly correlated with the MMP collections by week, P < .05???????????????.??????80 1 I. INTRODUCTION Tires have long been recognized as important sites for mosquito development. The ability of discarded tires to hold and maintain water allows for successful larval habitats for many mosquito species to develop. Because of this ability tire dumps promote the proliferation and dispersal of mosquitoes, increasing the potential of public health problems ranging from nuisance complaints to transmission of mosquito-borne diseases. Baumgartner (1988) stated that the ?tire problem? is magnified because tire- breeding mosquitoes develop rapidly, enhancing their vector ability. The most notable tire-breeding mosquito species of medical importance in the southeastern United States are Aedes albopictus, Ae. aegypti, Ochlerotatus triseriatus, and Oc. japonicus. Each of these species is a competent vector for more than one important disease of humans. Aedes albopictus, an Asian species introduced into the continental U.S. in 1985 (Francy et al. 1990) via tire shipments from Asia (Spenger and Wuithiranyagool 1986), has established itself in the eastern half of the U. S. (Moore and Mitchell 1997, Moore 1999). Aedes albopictus is a competent vector of Dengue and Yellow Fever Viruses (Gokhale et al. 2001). Laboratory tests have shown that Ae. albopictus is a competent vector of over 22 arboviruses (Moore and Mitchell 1997) including Ross River Virus and West Nile Virus (Mitchell et al. 1987). 2 Eastern Equine Encephalitis Virus (Moore and Mitchell 1997) and La Crosse Virus (Erwin et al. 2002) has been isolated from field-collected specimens. This mosquito breeds in a variety of natural and artificial containers. Aedes aegypti, introduced into the Americas via the slave trade, is the primary vector of Yellow Fever Virus (Fontenille et al. 1997). This species is also a competent vector of Dengue Virus (Thavara et al. 2001). In the 1960s, Ae. aegypti dispersal in the continental United States was linked to interstate tire shipments (Haverfield and Hoffman 1966). This mosquito breeds in a variety of natural and artificial containers. Ochlerotatus triseriatus, the primary vector of La Crosse Encephalitis Virus in the upper Midwest (DeFoliart et al. 1986), is widely distributed across the eastern half of the U.S. and southern Canada (Darsie and Ward 1981). The number of La Crosse cases has been directly linked to the number of discarded tries containing Oc. triseriatus (Beier et al. 1982). This species breeds primarily in water-filled cavities in deciduous trees, but also in artificial containers such as tires (Joy et al. 2003). Ochlerotatus japonicus, a native of Japan and Korea (Tanaka et al. 1979), was first detected in America in New York in 1998 (Peyton et al. 1999). Peyton et al (1999) suggested that the mode of introduction into the northeastern United States was in used tires. This mosquito has recently extended its southern range into Georgia, North Carolina, and Alabama (Gray et al. 2005 and Mullen 2005). Ochlerotatus japonicus is the primary vector of Japanese Encephalitis Virus in its native range. This species is an efficient laboratory vector of West Nile Virus (Sardelis and Turell 2001), La Crosse Virus (Sardelis et al. 2002), and Eastern Equine Encephalitis Virus (Sardelis et al. 2002). West Nile Virus has been detected in multiple pools of Oc. japonicus collected in the 3 northeastern United States during the summer of 2000 (Centers for Disease Control and Prevention 2000). This species is a container-breeder, utilizing both natural and artificial containers (Scott et al. 2001). Since many medically important mosquito species utilize tires as larval habitats, it is important to know which mosquitoes are breeding in tires in urban settings. One way to determine the mosquito species breeding in tires is by sampling tires for mosquito larvae. Another method involves collecting adults using various trapping methods and attractants. Many tire-breeding mosquitoes are difficult to capture using standard mosquito trapping devices such as the CDC light trap and gravid trap. In recent years octenol, a chemical widely used to attract blood-feeding insects, has been used to enhance mosquito collections. Octenol has not been evaluated for enhancing collections of tire-breeding mosquitoes. The response of Ae. albopictus to octenol is of particular interest because of its large populations in urban areas and its vectorial capacity for mosquito-borne viruses. In 2004 and 2005 field work was conducted in Alabama to survey and monitor tire- breeding mosquito populations in urban settings. Discarded-tire sites were sampled to determine species breeding in tire in Alabama, species distribution by county, the extent of Ae. aegypti displacement in tires, the seasonal occurrence of tire-breeding mosquitoes in Alabama, and the correlation of seasonal occurrence of mosquitoes by physiographic region in Alabama. Adult populations were also monitored at three field sties at Auburn and Phenix City, Alabama to determine if octenol enhances collections of tire-breeding mosquitoes. 4 Literature Cited Baumgartner DL. 1988. Suburban accumulations of discarded tires in northeastern Illinois and their associated mosquitoes. J Am Mosq Control Assoc 4:500-508. Beier JC, Travis M, Patricoski C, Kranzfelder J. 1983. Habitat segregation among larval mosquitoes (Diptera: Culicidae) in tire yards in Indiana, USA. J Med Entomol 20:76-80. Centers for Disease Control and Prevention. 2000. Update: West Nile virus activity- eastern United States, 2000. MMWR 49:1044-1047. Darsie RF Jr, Ward RA. 1981. Identification and Geographical Distribution of the Mosquitoes of North America, North of Mexico. Salt Lake City, UT: American Mosquito Control Association. DeFoliart GR, Watts DM, Grimstad PR. 1986. Changing patterns of mosquito-borne arboviruses. J Am Mosq Control Assoc 2:437-455. Erwin PC, Jones TF, Gerhardt RR, Halford SK, Smith AB, Patterson LER, Gottfried KL, Burkhalter KL, Nasci RS, Schaffner W. 2002. La Crosse encephalitis in Eastern Tennessee: clinical, environmental, and entomological characteristics from a blinded cohort study. Am J of Epidemiology 155:1060-1065. Fontenille DL, Lochouarn ND, Sokhna C, Lemasson JJ. 1997. High annual and seasonal variations in malaria transmission by anophelines and vector species composition in Dielmo, a holoendemic area in Senegal. Am J Trop med Hyg 56:247-253. Francy DB, Karabatsos N, Wesson DM, Moore CG, Lazuick JS Jr, Niebylski ML, Tsai TF, Craig GB Jr. 1990. A new arbovirus from Aedes albopictus, an Asian mosquito established in the United States. Science 250:1738-1740. 5 Grimstad PR, Haramis LD. 1984. Aedes triseriatus (Diptera: Culicidae) and La Crosse virus III: Enhanced oral transmission by nutrition deprived mosquitoes. J Med Entomol 21:249-256. Joy JE, Hanna AA, Kennedy BA. 2003. Spatial and temporal variation in the mosquitoes (Diptera: Culicidae) inhabiting waste tires in Nicholas County, West Virginia. J Med Entomol 40:73-77. Mitchell CJ, Miller BR, Gubler DJ. 1987. Vector competence of Aedes albopictus from Houston, Texas, for dengue serotypes 1 to 4, Yellow Fever and Ross River viruses. J Am Mosq Control Assoc 3:460-465. Moore CG. 1999 Aedes albopictus in the United States: current status and prospects for further spread. J Am Mosq Control Assoc 15:221-227. Moore CG, Mitchell CJ. 1997. Aedes albopictus in the United States: ten-year presence and public health implications. Emerg Infec Dis 3:329-334. Mullen GR. 2005. First report of Ochlerotatus japonicus in Alabama. Alabama Vector Management Society Newsletter 15 (2):2. Peyton EL, Campbell SR, Candeletti TM, Romanowski M, Crans JW. 1999. Aedes (Finlaya) japonicus japonicus (Theobald), a new introduction into the United States. J Am Mosq Control Assoc 15:238-241. Sardelis MR, Dohm DJ, Pagac B, Andre RG, Turell MJ. 2002. Experimental transmission of Eastern Equine Encephalitis virus by Ochlerotatus j. japonicus (Diptera: Culicidae). J Med Entomol 39:480-484. Sardelis MR, Turell MJ. 2001. Ochlerotatus j. japonicus in Frederick County, Maryland: discovery, distribution, and vector competence for West Nile virus. J Am Mosq Control Assoc 17:137-141. Scott JJ, Carle FL, Crans JW. 2001. Ochlerotatus japonicus collected from natural rockpools in New Jersey. J Am Mosq Control Assoc 17:91-92. 6 Sprenger D, Wuithiranyagool T. 1986. The discovery and distribution of Aedes albopictus in Harris County, Texas. J Am Mosq Control Assoc 2:217-218. Tanaka K, Mizusawa K, Saugstad ES. 1979. A revision of the adult and larval mosquitoes of Japan (including the Ryukyu Archipelago and the Ogasawara Islands) and Korea (Diptera: Culicidae). Contrib Am Entomol Inst (Ann Arbor) 16: 1-987. Thavara U, Tawtsin A, Chansang C, Kong-ngamsuk W, Paosriwong S, Boon-Long J, Rongsriyam Y, Komalamisra N. 2001. Larval occurrence, oviposition behavior and biting activity of potential mosquito vectors of dengue on Samui Island, Thailand. J Vec Ecology 26:172-180. Thompson WH, Anslow RO, Hanson RP, DeFoliart GR. 1972. La Crosse virus isolations from mosquitoes in Wisconsin, 1964-1968. Am J Trop Med Hyg 23:694-700. 7 II. STATE-WIDE SURVEY OF TIRE-BREEDING MOSQUITOES (DIPTERA: CULICIDAE) IN ALABAMA Objectives 1. To determine the species of mosquitoes breeding in tires in Alabama. 2. To determine the species distribution by county throughout the State. 3. To determine the extent to which Aedes albopictus has displaced Aedes aegypti as the dominant tire-breeding mosquito in Alabama. 4. To determine the seasonal occurrence of mosquitoes breeding in tires in Alabama. 5. To determine if there is a correlation between seasonal occurrence of tire-breeding mosquitoes and the physiographic regions of Alabama. In order to effectively apply mosquito-control measures, the diversity of mosquito larval habitats must be understood. Tires are just one of many larval development habitats that provide sufficient nutrients that allow mosquitoes to flourish. Over the years studies have focused on tires as mosquito larval habitats. The importation of mosquito larvae in tires was first reported in the mid 1940s. Pratt et al. (1946) reported that shiploads of tires arriving from Asian ports were heavily 8 infested with seven mosquito species including Ae. albopictus. Haverfield and Hoffman (1966) investigated intrastate movement of tire shipments in Texas and associated that movement with the dispersal of Ae. aegypti across the state. Other studies, including Beier et al. (1983), focused on identifying the ecological factors that regulate mosquito production in tires. Studies have also investigated the likelihood that natural container-breeding mosquitoes, such as Ochlerotatus triseriatus and Oc. atropalpus, would oviposit in tires (Haramis 1984, Restifo and Lanzaro 1980, Berry and Craig 1984). These studies concluded that both Oc. triseriatus and Oc. atropalpus readily oviposit in tires, potentially altering their development time and their vectorial capacity (Haramis 1984, Baumgartner 1988). However, after the introduction and establishment of Ae. albopictus in the United States (1985) via the tire trade (Sprenger and Wuithiranyagool 1986), attention has shifted to recognizing the importance of tires as a means of introduction of nonindigenous mosquito species. Reiter and Sprenger (1987) investigated the tire trade and found that just after the initial discovery of Ae. albopictus in Texas, 12 other states were infested with Ae. albopictus populations. Nearly all of the infestations were in tires. With the ever increasing human population and accumulation of used tires in urban areas, along with the introduction of Ae. albopictus, focus has again shifted to recognizing the importance of discarded tires as a major source of mosquito production. Many studies have been conducted to determine what mosquito species are breeding in tires. A study in Connecticut (1988) investigated nine tire disposal sites to determine the mosquito species present with specific interest in Ae. albopictus (Andreadis 1987). Aedes 9 albopictus at that time had not yet extended its distribution into Connecticut, but had been found in surrounding states Maryland and Delaware (Centers for Disease Control and Prevention 1987). Another study in Illinois investigated the species composition and abundance of mosquito larvae at a large tire dump site where Ae. albopictus had previously been reported (Lampman et al. 1997). Other studies have focused on characterizing tires habitats, to determine factors influencing mosquito production (Morris and Robinson 1994, Joy Hildreth-Whitehair 2000, Joy et al. 2003). From these studies 18 mosquito species have been reportedly collected from tires in the eastern half of the United States. These species are Ae. albopictus, Ae. aegypti, Ae. vexans, Anopheles barberi, An. punctipennis, An. quadrimaculatus, Culex pipiens, Cx. quinquefasciatus, Cx. restuans, Cx. salinarius, Cx. territans, Culiseta melanura, Ochlerotatus atropalpus, Oc. bahamensis, Oc. triseriatus, Oc. japonicus, Orthopodomyia signifera, and Toxorhynchites rutilus. See Appendix for a list of the mosquito species and their respective authors mentioned throughout the text. Sixteen of the above 18 mosquito species occur in Alabama, the exceptions being Cx. pipiens and Oc. bahamensis (Darsie and Ward 2005). Culex pipiens is widely distributed across the northern United States and in British Columbia, Canada. Ochlerotatus bahamensis distribution is localized to only the southeastern portion of Florida. The first detection of Ae. albopictus in the state of Alabama was from a tire site in Cullman County in 1986. There has never been a survey of tire-breeding mosquitoes in Alabama prior to, and since, the introduction of Ae. albopictus. This survey was conducted to determine what mosquito species are breeding in tires in Alabama. Aedes 10 aegypti was once the dominant mosquito breeding in tree holes and artificial containers, including tires in Alabama. Recent studies have suggested changes in species composition and dominance structure (Edgerly et al. 1993) since the establishment of Ae. albopictus in North America. Aedes albopictus has been associated with the displacement of native container-breeding mosquitoes, such as Ae. aegypti and Oc. triseriatus (Juliano 1998). In the last 17 years, Ae. aegypti has been scarce or absent in Alabama (G. R. Mullen unpublished). One objective of this survey was to determine to the abundance of Ae. aegypti in tires in Alabama. This is one of the few comprehensive state-wide surveys of tire-breeding mosquitoes that has been conducted in the United States. In Florida Ae. albopictus movement has been documented since its first detection in 1986. In 1992 Ae. albopictus was collected in 64 out of 67 counties in Florida from containers such as tires and cemetery vases (O?Meara et al. 1993) Another comprehensive study was conducted in West Virginia over a six-year period. Containers, including tires, from 54 of 55 West Virginian counties were sampled for container-breeding mosquitoes (Joy and Hildreth- Whitehair 2000). These data were correlated with the population size of Oc. triseriatus and the incidence of La Crosse Virus in the counties sampled. Seasonal data of tire-breeding mosquitoes generated by this survey will be useful for mosquito-control programs throughout the State and in determining if there is any correlation between the seasonal occurrence of tire-breeding mosquitoes and the different physiographic regions of Alabama. Because temperature directly affects developmental time, it is possible that seasonal differences in the emergence of tire-breeding mosquitoes may occur in these five physiographic regions. 11 Materials and Methods This study was organized by coordinating the Alabama Department of Public Health area administrators and county environmentalists to sample discarded tires in their respective counties from May 1 to October 31, 2004 (Figure 1). This was followed by sampling tires in 2005 in counties in which tire-breeding mosquitoes were not collected during the summer of 2004. The tire sites sampled ranged from two tires per tire site to huge tire sites with over 50,000 tires. In 2004 two or more tire sites per county were sampled twice a month, one collection taken during the first half of the month and the second collection during the latter half of the month. One or two tire sites were sampled from the remaining counties during 2005. Each ACHD county environmentalist was asked to follow the same sampling protocol: (1) agitate the tire before collecting a sample to ensure that both bottom-feeding and top-feeding larvae were sampled, (2) collect a sample using either a dipper or baster, (3) filter the sample through a small aquarium net, (4) invert the aquarium net into a small container of alcohol, and (5) pipette the larvae from the small container of alcohol into a four-dram vial of 70% ethyl alcohol. At least four tires were sampled from each tire site, with a minimum collection of 20 larvae per collection site. Larvae were sent to the medical entomology lab at Auburn University via the Alabama Department of Public Health Courier system to be identified. All third- and fourth-instar larvae were identified using Darsie and Ward?s (1981) Identification and 12 Geographical Distribution of the Mosquitoes of North America, North of Mexico. First- instar larvae, second-instar larvae, and pupae were not identified during this survey. Voucher specimens for each species have been deposited in the Auburn University Insect Collection. The data generated from this survey were used to determine if there was a correlation between the physiographic regions in Alabama (Figure 2) and seasonal occurrence of tire-breeding mosquitoes in these regions. The five physiographic regions are: Highland Rim, Cumberland Plateau, Alabama Valley and Ridge, Piedmont Upland, and the East Gulf Coastal Plain. Soils in the Highland Rim region are mostly red clay of limestone origin. The forests are comprised mostly of hardwoods, red cedar, and some pines. The Cumberland Plateau has mostly sandy loams, although clay soils are not uncommon in the southern portion of this region. The Alabama Valley and Ridge has soils ranging from gravelly loams to clay where the Piedmont Upland has clay soils that tend to be rocky. The East Gulf Coastal Plain soils vary from acid sands and sandy loams to heavy, calcareous, alkaline types. (Mount 1975) Results Tires were sampled in 52 of 67 counties from May 1 to October 31, 2004 (Figure 3a). The 15 counties in which collections were not made in 2004 were: Cherokee, Coosa, Houston, Jefferson, Lamar, Lawrence, Limestone, Madison, Marengo, Perry, Randolph, Tallapoosa, Walker, Wilcox, and Winston, (Figure 3b). Tires were sampled from the counties that did not collect larval samples in 2004 during the summer of 2005. 13 Houston County was sampled in April 2005; samples were collected from Cherokee, Jefferson, Lamar, Lawrence, Limestone, Madison, Randolph , Tallapoosa ,Walker, and Winston in July; and Coosa, Marengo, Perry, and Wilcox counties in August. County environmentalist in Jefferson and Houston Counties collected larval samples in their respective counties in 2005. The 13 remaining counties were sampled by going myself to each county and locating a tire site. A total of 169 tire sites was sampled from throughout this survey (Figure 4) with a total of 13,022 mosquito larvae identified, representing 12 mosquito species in 7 genera (Table 1). The most frequently collected mosquito species were Ae. albopictus (71%), Cx. territans (7.5%), and Oc. triseriatus (7.1%). The following species were also collected: Cx. restuans (5.2%), Cx. salinarius (3.5%), Or. signifera (2.7%), Cx. quinquefasciatus (1.2%), Oc. atropalpus (<1%), Tx. rutilus (<1%), An. punctipennis (<1%), An. quadrimaculatus (<1%), An. spp. (<1%), and Ps. columbiae (<1%). Total larval collections throughout this survey by county and month are shown in Table 2. The geographic distribution, by county, of each mosquito species collected in this survey from tires sites is presented in Figures 5-8. Aedes albopictus was collected from all 169 tire sites sampled, indicating that it is established in every county in Alabama. Aedes albopictus overlapped in tire yards with Oc. triseriatus in 39 counties in Alabama. No Ae. aegypti larvae were found in tires during this study, nor larvae of the recently introduced Oc. japonicus. There was no difference in the seasonality of Ae. albopictus and Oc. triseriatus by 2-week intervals observed in tires during this survey (Figure 9). There was a seasonal difference observed in collections of Oc. atropalpus, Ps. columbiae, and Anopheles 14 species. Ochlerotatus atropalpus was collected first during the first half of June and then was not collected again until the second half of July. Psorophora columbiae was only collected during the last half of June and the month of July. Anopheles species were only collected from tires during the first part of the summer months. The remaining species were collected consistently throughout each collection period, i.e., first half or second half of months May-October, 2004 and 2005 (Table 3). The data were not sufficient to show any correlation between physiographic region and seasonal occurrence of tire-breeding mosquitoes (Tables 4 and 5). Anopheles species, Cx. quinquefasciatus, Cx. salinarius, Oc. atropalpus, Or. signifera, and Ps. columbiae larvae were not collected in the Piedmont region in Alabama throughout this survey. However, distributions of these species fall within the Piedmont region according to Darsie and Ward (2005). Anopheles quadrimaculatus, Oc. atropalpus, and Ps. columbiae larvae were not collected in the Cumberland Plateau or Highland Rim regions, although the reported distributions of these species fall within these regions (Darsie and Ward 2005) (Figure 10). Discussion Throughout this survey tire dump sites, service stations, tire dealers, auto repair shops, and salvage yards were sampled in rural and urban areas of Alabama. All of these sites produced mosquitoes suggesting that the ?tire problem? (Baumgartner 1988) is not just localized to large tire dump sites in rural areas. A total of 13 mosquito species was collected from tires in Alabama. Many of the urban tire sites sampled had more than one species of mosquito present throughout the entire survey. 15 Aedes albopictus was collected from tire sites in combination with the 12 other mosquito species collected throughout the survey. Culex territans was frequently collected from the same tire sites as Cx. restuans and Oc. triseriatus. Although previous studies had reported that Cx. territans does not commonly utilize tires as a larval habitat (Wilmot et al. 1992, Jamieson et al. 1994), Joy et al. (2003) found relatively high occurrence of Cx. territans larvae in abandoned tire sites in their survey in West Virginia. Our data supports Joy et al.?s findings in that Cx. territans was the second most frequently collected tire-breeding species in Alabama throughout the survey. The distributions of 12 of the 13 mosquito species fell within their previously known ranges (Darsie and Ward 2005). However, collections of Oc. atropalpus occurred farther south, in Montgomery County, than its distribution shown in Darsie and Ward (2005). Craig (1980) suggested that once Oc. atropalpus was introduced into tire yards by both local and interstate transportation of discarded tires, this species would probably extend its range. Since Oc. atropalpus females are autogenous for their first ovarian cycle (O?Meara and Krasnick 1970), they can exploit large numbers of tires, in turn extending their distribution (Beier et al 1983). Ochlerotatus atropalpus was collected from only one tire site (Montgomery County) on one occasion. This does not necessarily confirm that Oc. atropalpus has extended its southern range in Alabama as this sample could have been collected from a tire recently shipped from an area where Oc. atropalpus populations are known to occur. The collections throughout this survey were inconsistent. Many counties did not collect from more than one tire site. Other counties only sampled during one month of the survey period. On the other hand, some counties were very consistent with their larval 16 collections, collecting from more than one tire site throughout the survey. Because of this no conclusions on the seasonality of tire-breeding mosquitoes in Alabama can be made from the data obtained. Psorophora columbiae was collected from only one tire site (Pickens County) in June and July 2004. This species usually breeds in woodland pools and has not been previously reported to breed in tires. This collection is presumed to be incidental and does not suggest that Ps. columbiae was regularly present in tires during the survey period. There were no collections of Oc. japonicus throughout this survey, nor had this species been reported in Alabama prior to this survey. The previously reported southern- most collection of this species was in Fulton County, Georgia (Gray et al. 2005). However, in the summer of 2005 a single adult Oc. japonicus female turned up in a CDC gravid trap in Jackson County, AL (Mullen 2005). Aedes albopictus was the only mosquito collected in the larval stage from tire sites in Jackson County during the tire- breeding mosquito survey in Alabama in 2004. Aedes albopictus was collected at every tire site sampled in this survey. Apparently this species has been successful at establishing itself throughout Alabama. Since Ae. aegypti was not collected at all in this survey, the implication is that Ae. albopictus has Ae. aegypti as the dominant tire-breeder in Alabama. This has similarly been observed by others, most notably in some habitats in Florida (O?Meara et al. 1992), South Carolina (Richardson et al. 1995), and Louisiana (Nasci 1995). Ochlerotatus triseriatus overlapped with Ae. albopictus in 39 of the 67 counties sampled. Previous studies have suggested that Ae. albopictus would displace Oc. 17 triseriatus because of its competitive advantage in both larval development time and its ability to hatch in high densities (Ho et al. 1989, Edgerly et al. 1993). However, Moore (1999) stated that temporal and spatial differences between these two species would decrease the likelihood of Oc. triseriatus being displaced by Ae. albopictus. The data in this survey showed that many natural container-breeders like Oc. triseriatus and Or. signifera readily oviposited in tires at the same sites in which large collections of Ae. albopictus were collected. This may be attributed to the fact that urbanization has reduced natural breeding sites, directly affecting breeding habits of certain tree-hole breeding mosquitoes. Since Ae. albopictus is still considered a potential vector in West Nile Virus transmission and is considered the number one nuisance mosquito in many urban areas of the southeastern United States, its distribution is relevant in assessing the potential public health risks. Because the public is aware of West Nile Virus, better strategies need to be implemented to control the ?tire problem?. This is already apparent in some of the counties in Alabama that have their own vector control units or mosquito control programs. In the cities of these counties, there is rapid turnover of tires. The tires are removed in a timely fashion so that they are not producing large populations of mosquitoes. Thus, educating the public on the importance that tires play in the production of mosquitoes can influence action to clean up tire sites. This study provides a baseline for other invasive mosquitoes that might become established in Alabama. Even though Ae. aegypti once considered to be the dominant tire-breeding mosquito in Alabama, we have no reported baseline for its distribution in tires in Alabama. With the recent detection of Oc. japonicus in Alabama, there is the 18 possibility of this introduced species becoming established in the state. Since Oc. japonicus readily develops in tires, the geographic spread of this species and its possible impact on Alabama?s tire-breeding mosquito fauna can be monitored in the future based on the baseline information provided. 19 Literature Cited Alto BW, Julian SA. 2001. Precipitation and temperature effects on populations of Aedes albopictus (Diptera: Culicidae): implication for range expansion. J Med Entomol 38:646-656. Andreadis TG. 1988. A survey of mosquitoes breeding in used tire stockpiles in Connecticut. J Am Mosq Control Assoc 4:256-260. Baumgartner DL. 1988. Suburban accumulations of discarded tires in northeastern Illinois and their associated mosquitoes. J Am Mosq Control Assoc 4:500-508. Beier JC, Travis M, Patricoski C, and Kranzfelder J. 1983. Habitat segregation among larval mosquitoes (Diptera: Culicidae) in tire yards in Indiana, USA. J Med Entomol 20:76-80. Centers for Disease Control and Prevention. 1987. Aedes albopictus infestation?United States. MMWR 36:769-773. Craig, GB Jr. 1980. Aedes atropalpus introduced into Indiana: Is this mosquito a potential vector of arboviruses? Proc Ind Vector Control Assoc 4:3-7. Darsie RF Jr., Ward RA. 1981. Identification and Geographical Distribution of the Mosquitoes of North America, North of Mexico. Salt Lake City, UT: American Mosquito Control Association. Darsie RF Jr., Ward RA. 2005. Identification and Geographical Distribution of the Mosquitoes of North America, North of Mexico. Gainesville, FL: American Mosquito Control Association. Eads RB. 1976. An unusual larval habitat for Culiseta melanura. Mosq News 36:544. Edgerly JS, Willey MS, Livdahl TP. 1993. The community ecology of Aedes egg hatching: implications for a mosquito invasion. Ecol Entomol 18:123-128. 20 Edgerly JS, Willey MS, Livdahl TP. 1999. Intraguild predation among larval treehole mosquitoes, Aedes albopictus, Ae. aegypti, and Ae. triseriatus (Diptera: Culicidae), in laboratory microcosms. J Med Entomol 36:394-399. Gray EW, Harrison BA, Womack ML, Kerce J, Neely CJ, Noblet R. 2005. Ochlerotatus japonicus japonicus (Theobald) in Georgia and North Carolina. J Am Mosq Control Assoc 21:144-146. Haramis LD. 1984. Aedes triseriatus: a comparison of density in tree holes vs. discarded tires. Mosq News 44:485-489. Haverfield LE, Hoffman BI. 1966. Used tires as a means of dispersal of Aedes aegypti in Texas. Mosq News 26:433-435. Ho BC, Ewert A, Chew L. 1989. Interspecific competition among Aedes aegypti, Ae. albopictus, and Ae. triseriatus (Diptera: Culicidae): larval development in mixed cultures. J Med Entomol 26:615-623. Jamieson, DH, Olson LA, Wilhide JD. 1994. A larval mosquito survey in northeastern Arkansas including a new record for Aedes albopictus. J Am Mosq Control Assoc 10:236-239. Juliano SA. 1988. Species introduction and replacement among mosquitoes: interspecific resource competition or apparent competition? Ecology 79:255- 268. Joy JE, Hildreth-Whitehair A. 2000. Larval habitat characterization for Aedes triseriatus (Say), the mosquito vector of La Crosse encephalitis in West Virginia. Wild Environ Med 11:79-83. Joy JE, Hanna AA, Kennedy BA. 2003. Spatial and temporal variation in the mosquitoes (Diptera: Culicidae) inhabiting waste tires in Nicholas County, West Virginia. J Med Entomol 40:73-77. 21 Lampman RS, Hanson S, Novak R. 1997. Seasonal abundance and distribution of mosquitoes at a rural waste tire site in Illinois. J Am Mosq Control Assoc 13:193-200. Moore CG. 1999. Aedes albopictus in the United States: current status and prospects for further spread. J Am Mosq Control Assoc 15:221-227. Morris CD, Robinson JW. 1994. Distribution of mosquito larvae in waste tire pile in Florida--an initial study. J Am Mosq Control Assoc 10:339-343. Mount RH. 1975. The Reptiles and Amphibians of Alabama. Auburn, AL. Auburn Printing Company. Mullen GR. 2005. First report of Ochlerotatus japonicus in Alabama. Alabama Vector Management Society Newsletter. 15 (2):2. Novak RJ, Steinly BA, Webb DW, Haramis L, Clarke J, Farmer B, and Cieslik R. 1990 Penetration rate of two pesticide carriers at a large used-tire storage facility in Chicago, Illinois. J Am Mosq Control Assoc 13:193-200. O?Meara, GF and Karsnick GJ. 1970. Dietary and genetic control of the expression of autogenous reproduction in Aedes atropalpus (Coq.) (Diptera: Culicidae). J Med Entomol 7:328-324. O?Meara GF, Evans LF, Gettman AD, and Cuda JP. 1995. Spread of Aedes albopictus and decline of Ae. aegypti (Diptera: Culicidae) in Florida. J Med Entomol 32:554-562. O?Meara GF, Gettman AD, Evans LF, Jr, Curtis GA. 1993. The spread of Aedes albopictus in Florida. American Entomologist 39:163-172. O?Meara GF, Gettman AD, Evans LF, Scheel FD. 1992. Invasion of cemeteries in Florida by Aedes albopictus. J Am Mosq Control Assoc 8: 1-10. 22 Pratt JJ JR, Heterick RH, Harrison JB, Haber L. 1946. Tires as a factor in the transportation of mosquitoes by ships. Mil Surgeon 99:785-788. Reiter P, Sprenger D. 1987. The used tire trade: a mechanism for the worldwide dispersal of container breeding mosquitoes. J Am Mosq Control Assoc 3:494- 501. Restifo RA, Lanzaro GC. 1980. The occurrence of Aedes atropalpus (Coquillett) breeding in tires in Ohio and Indiana. Mosq News 40:292-294. Sprenger D, Wuithiranyagool T. 1986. The discovery and distribution of Aedes albopictus in Harris County, Texas. J Am Mosq Control Assoc 2: 217-218. Wilmot TR, Zeller DS, Merritt RW. 1992. A key to container-breeding mosquitoes of Michigan (Diptera: Culicidae), with notes on their biology. The Great Lakes Entomol 25:137-148. 23 Table 1. Total numbers and percentages of mosquito species collected from tires in Alabama by month, based on the 2004-2005 larval survey. Species are listed in descending order of the total numbers of each species collected. Mosquito Species May June July Aug. Sept. Oct. Totals % Aedes albopictus 129 1832 3234 2088 1451 429 9163 71 Culex territans 29 135 171 296 220 192 1043 7.5 Ochlerotatus triseriatus 0 190 370 195 148 18 921 7.1 Culex restuans 44 393 94 156 62 28 777 5.2 Culex salinarius 15 58 121 61 85 13 353 3.5 Orthopodomyia signifera 0 51 132 32 73 28 316 2.7 Culex quinquefasciatus 3 9 27 8 93 36 176 1.2 Ochlerotatus atropalpus 0 35 39 30 29 0 133 0.9 Toxorhynchites rutilus 0 14 28 22 12 7 83 0.7 Anopheles spp. 1 3 10 8 0 1 23 0.2 Psorophora columbiae 0 13 8 0 0 0 21 0.2 Anopheles punctipennis 0 10 0 0 0 0 10 < 0.1 Anopheles quadrimaculatus 0 3 0 0 0 0 3 < 0.1 Totals 221 2746 4234 2896 2173 752 13022 100 24 T a bl e 2. T o t a l n u m b er s o f m o s q u i t o s p ec i e s co l l e c t ed by co un t y a n d m o n t h f r o m t i r e s i n Al a b a m a, b a s e d o n t h e 2004- 2005 l a r v a l s u r v ey . T h e s i x t y - s e v e n A l ab am a c o u n t i es a r e l i s t e d i n al ph ab e t i cal or d e r . M o nt h Co un t y M o s qui t o S p e c i e s M a y J u n e J u l y A ugus t S e pt e m b e r O c t o b e r Au t a u g a Ae de s albopic t us 58 160 31 52 C u le x te rrit ans 1 Oc hle r otatus trise r ia tus 12 20 C u le x re stuans 13 4 Cule x sali narius 4 Orthopodomy i a s i gnife ra 11 6 C u le x quinque fa sc iatus 5 Tox o rhy n c h ite s ru tilus 2 2 Anophe le s quadrimac ul atus 2 To t a l s 0 74 190 37 82 0 Ba l d w i n Ae de s albopic t us 9 33 14 1 1 C u le x te rrit ans 1 4 Oc hle r otatus trise r ia tus 1 3 2 C u le x re stuans Cule x sali narius 1 13 Orthopodomy i a s i gnife ra 5 5 To t a l s 0 11 55 20 6 1 Ba r bou r Ae de s albopic t us 50 117 209 32 C u le x te rrit ans 5 Oc hle r otatus trise r ia tus 2 5 25 C u le x re stuans 20 Cule x sali narius 5 3 Orthopodomy i a s i gnife ra 5 To t a l s 0 70 134 217 0 32 B i bb Ae de s albopic t us 20 38 C u le x te rrit ans Oc hle r otatus trise r ia tus 6 11 Oc hle r otatus a t ropalpu s 17 To t a l s 0 43 49 0 0 0 Bl ou n t Ae de s albopic t us 20 Oc hle r otatus trise r ia tus 11 C u le x re stuans 8 Anophe le s quadrimac ul atus 2 To t a l s 0 41 0 0 0 0 B u llo c k Ae de s albopic t us 60 To t a l s 0 60 0 0 0 0 B u tle r Ae de s albopic t us 5 38 68 62 5 C u le x te rrit ans 4 1 10 1 C u le x re stuans 6 5 5 Cule x sali narius 1 C u le x quinque fa sc iatus 11 5 To t a l s 0 15 56 83 62 11 26 C a l h oun Ae de s albopic t us 57 44 33 78 30 Oc hle r otatus trise r ia tus 1 3 Cule x sali narius 11 7 To t a l s 0 58 58 33 85 30 Cha m b e r s Ae de s albopic t us 26 18 C u le x te rrit ans 5 Oc hle r otatus trise r ia tus 8 To t a l s 0 0 26 31 0 0 C h il to n Ae de s albopic t us 11 116 29 26 7 C u le x te rrit ans 11 Oc hle r otatus trise r ia tus 10 4 9 C u le x re stuans 7 Cule x sali narius 5 C u le x quinque fa sc iatus 2 To t a l s 0 21 116 40 37 23 C h e r o k ee Ae de s albopic t us 17 C u le x te rrit ans 15 C u le x re stuans 21 Oc hle r otatus a t ropalpu s 3 Anophe le s punc tipe nnis 3 Anophe le s quadrimac ul atus 2 To t a l s 0 61 0 0 0 0 27 Ch o c t a w Ae de s albopic t us 10 To t a l s 0 10 0 0 0 0 Cl a r ke Ae de s albopic t us 4 38 C u le x te rrit ans 4 Oc hle r otatus trise r ia tus 3 17 2 C u le x re stuans 25 Orthopodomy i a s i gnife ra 41 10 Tox o rhy n c h ite s ru tilus 5 3 Anophe le s spp . To t a l s 0 7 105 40 0 0 Cl a y Ae de s albopic t us 15 To t a l s 0 0 15 0 0 0 Cl e b urn e Ae de s albopic t us 7 91 56 38 C u le x te rrit ans 2 Oc hle r otatus trise r ia tus 5 15 3 1 To t a l s 0 12 106 59 41 0 C o f f ee Ae de s albopic t us 36 123 85 121 38 C u le x te rrit ans 23 Oc hle r otatus trise r ia tus 3 C u le x re stuans 7 1 Orthopodomy i a s i gnife ra 13 4 C u le x quinque fa sc iatus 1 To t a l s 0 49 137 85 122 62 28 C o lb e r t Ae de s albopic t us 42 26 17 2 C u le x te rrit ans 42 Oc hle r otatus trise r ia tus 2 Cule x sali narius 3 Orthopodomy i a s i gnife ra To t a l s 0 42 0 26 61 5 C o n ecu h Ae de s albopic t us 41 18 13 Oc hle r otatus trise r ia tus 5 3 8 To t a l s 0 0 46 21 21 0 Co os a Ae de s albopic t us 43 To t a l s 0 0 43 0 0 0 C o v i ng t o n Ae de s albopic t us 54 129 131 3 36 C u le x te rrit ans 3 Oc hle r otatus trise r ia tus 2 11 C u le x re stuans 1 Cule x sali narius 2 19 6 Orthopodomy i a s i gnife ra 2 4 To t a l s 0 61 131 151 3 57 Cr e n s h a w Ae de s albopic t us 82 To t a l s 0 0 0 82 0 0 29 C u ll ma n Ae de s albopic t us 27 71 31 47 C u le x te rrit ans 5 2 Oc hle r otatus trise r ia tus 12 12 2 C u le x re stuans 28 5 Cule x sali narius 8 C u le x quinque fa sc iatus 39 To t a l s 0 67 83 36 96 7 Da l e Ae de s albopic t us 80 109 179 135 144 21 C u le x te rrit ans 44 1 13 Oc hle r otatus trise r ia tus 5 28 35 19 32 Cule x sali narius 8 7 Orthopodomy i a s i gnife ra 14 14 20 5 C u le x quinque fa sc iatus 1 2 Tox o rhy n c h ite s ru tilus 1 1 9 7 Anophe le s punc tipe nnis 1 To t a l s 86 191 236 177 206 39 Da l l a s Ae de s albopic t us 22 C u le x re stuans 1 C u le x quinque fa sc iatus 3 Oc hle r otatus a t ropalpu s To t a l s 0 26 0 0 0 0 D e ka l b Ae de s albopic t us 34 48 To t a l s 0 0 34 48 0 0 El mo r e Ae de s albopic t us 98 83 30 To t a l s 0 98 130 0 0 0 Es c a mb ia Ae de s albopic t us 22 60 47 46 41 C u le x te rrit ans 15 14 Oc hle r otatus trise r ia tus 4 C u le x re stuans 8 Cule x sali narius 10 2 1 To t a l s 0 40 75 49 51 55 Eto w ah Ae de s albopic t us 3 50 2 16 C u le x te rrit ans 14 Oc hle r otatus trise r ia tus 13 C u le x re stuans 2 42 58 Cule x sali narius 4 Oc hle r otatus a t ropalpu s 12 32 30 29 Tox o rhy n c h ite s ru tilus 2 Anophe le s punc tipe nnis 1 To t a l s 0 17 97 72 93 31 F a y e tte Ae de s albopic t us 15 To t a l s 0 0 0 15 0 0 F r an k lin Ae de s albopic t us 2 2 5 To t a l s 2 2 5 0 0 0 Ge n e v a Ae de s albopic t us 30 16 Oc hle r otatus trise r ia tus 26 59 Cule x sali narius 3 5 To t a l s 0 59 80 0 0 0 31 G r een e Ae de s albopic t us 69 47 7 33 C u le x te rrit ans 1 Cule x sali narius 38 58 C u le x quinque fa sc iatus 2 Tox o rhy n c h ite s ru tilus 1 Anophe le s punc tipe nnis 3 1 To t a l s 0 70 88 8 94 0 Ha l e Ae de s albopic t us 55 106 C u le x te rrit ans 2 Cule x sali narius 3 To t a l s 0 55 106 5 0 0 He n r y Ae de s albopic t us 53 41 103 43 27 C u le x te rrit ans 18 Oc hle r otatus trise r ia tus 3 C u le x re stuans 31 Cule x sali narius 1 2 C u le x quinque fa sc iatus 8 Tox o rhy n c h ite s ru tilus 1 2 1 Anophe le s quadrimac ul atus 1 To t a l s 0 86 46 123 45 35 Ho u s t o n Ae de s albopic t us 38 C u le x te rrit ans 20 C u le x re stuans 24 C u le x quinque fa sc iatus 3 Anophe le s punc tipe nnis 1 32 To t a l s 86 0 0 0 0 0 Ja c k so n Ae de s albopic t us 55 To t a l s 0 55 0 0 0 0 Je f f e r so n Ae de s albopic t us 7 C u le x te rrit ans 2 Tox o rhy n c h ite s ru tilus 2 To t a l s 0 11 0 0 0 0 La m a r Ae de s albopic t us 12 C u le x te rrit ans 5 C u le x re stuans 23 Anophe le s punc tipe nnis 1 To t a l s 0 41 0 0 0 0 L a ude r d a l e Ae de s albopic t us 3 13 C u le x te rrit ans 4 15 93 85 Oc hle r otatus trise r ia tus 14 25 C u le x re stuans 2 Tox o rhy n c h ite s ru tilus 1 1 1 Anophe le s quadrimac ul atus 4 1 To t a l s 13 15 55 93 86 0 La w r en ce Ae de s albopic t us 46 C u le x re stuans 15 Cule x sali narius 5 Anophe le s punc tipe nnis 3 To t a l s 0 69 0 0 0 0 33 Lee Ae de s albopic t us 42 109 79 49 23 C u le x te rrit ans 5 2 13 Oc hle r otatus trise r ia tus 10 12 C u le x re stuans 23 Orthopodomy i a s i gnife ra 1 4 Tox o rhy n c h ite s ru tilus 1 1 To t a l s 0 54 121 107 51 41 L ime s t o n e Ae de s albopic t us 24 C u le x te rrit ans 5 Oc hle r otatus trise r ia tus 3 C u le x re stuans 22 Anophe le s punc tipe nnis 1 To t a l s 0 55 0 0 0 0 Lo w n d e s Ae de s albopic t us 3 C u le x te rrit ans 5 C u le x quinque fa sc iatus 1 To t a l s 0 0 0 9 0 0 Ma c o n Ae de s albopic t us 38 48 C u le x te rrit ans 2 Oc hle r otatus trise r ia tus 2 20 Cule x sali narius 3 Orthopodomy i a s i gnife ra 14 Tox o rhy n c h ite s ru tilus 2 6 34 To t a l s 0 0 0 45 84 6 Ma d i so n Ae de s albopic t us 31 C u le x re stuans 5 Cule x sali narius 1 C u le x quinque fa sc iatus 3 Anophe le s punc tipe nnis 3 To t a l s 0 43 0 0 0 0 Ma r e n g o Ae de s albopic t us 50 C u le x te rrit ans 43 Oc hle r otatus trise r ia tus 5 To t a l s 0 0 98 0 0 0 Ma r i o n Ae de s albopic t us 6 17 172 29 20 C u le x te rrit ans 4 16 Oc hle r otatus trise r ia tus 8 39 3 C u le x re stuans 5 3 C u le x quinque fa sc iatus 1 Tox o rhy n c h ite s ru tilus 1 To tal s 6 32 211 0 39 36 Ma r s h a l l Ae de s albopic t us 2 12 54 8 25 C u le x te rrit ans 2 3 Oc hle r otatus trise r ia tus 8 7 C u le x re stuans 1 22 1 1 Cule x sali narius 19 13 35 Orthopodomy i a s i gnife ra 7 5 C u le x quinque fa sc iatus 3 Tox o rhy n c h ite s ru tilus 5 To t a l s 3 43 87 8 57 0 M o b ile Ae de s albopic t us 46 45 37 66 63 C u le x te rrit ans 1 11 14 1 10 Oc hle r otatus trise r ia tus 18 2 11 7 C u le x re stuans 9 8 Cule x sali narius 6 4 Orthopodomy i a s i gnife ra 8 1 C u le x quinque fa sc iatus 3 11 7 5 23 Tox o rhy n c h ite s ru tilus 2 10 2 3 To ta ls 0 93 81 62 86 112 Mo n r o e Ae de s albopic t us 82 66 33 C u le x te rrit ans 27 4 Oc hle r otatus trise r ia tus 1 Cule x sali narius 10 Orthopodomy i a s i gnife ra 7 19 To tal s 0 0 92 100 57 0 M o n t go m e r y Ae de s albopic t us 144 107 66 94 2 C u le x te rrit ans 13 22 12 44 Oc hle r otatus trise r ia tus 1 39 46 16 C u le x re stuans 5 Cule x sali narius 1 9 36 Orthopodomy i a s i gnife ra 1 21 4 Oc hle r otatus a t ropalpu s 7 Tox o rhy n c h ite s ru tilus 3 3 To t a l s 0 160 199 136 154 11 Mo r g a n Ae de s albopic t us 3 C u le x te rrit ans 8 C u le x re stuans 16 Cule x sali narius 6 C u le x quinque fa sc iatus 7 To t a l s 0 40 0 0 0 0 Pe r r y Ae de s albopic t us 38 C u le x te rrit ans 22 Oc hle r otatus trise r ia tus 7 To t a l s 0 0 67 0 0 0 P i ck en s Ae de s albopic t us 88 247 120 46 21 C u le x te rrit ans 15 12 91 25 75 Oc hle r otatus trise r ia tus 1 17 5 5 C u le x re stuans 24 12 47 Cule x sali narius 21 Orthopodomy i a s i gnife ra 1 C u le x quinque fa sc iatus 20 Tox o rhy n c h ite s ru tilus 2 P s orophora c o lumbiae 13 8 Anophe le s punc tipe nnis 2 4 Anophe le s quadrimac ul atus To t a l s 0 142 318 288 78 96 37 P i ke Ae de s albopic t us 16 18 30 To t a l s 0 16 18 30 0 0 Ra n d ol ph Ae de s albopic t us 31 C u le x te rrit ans 1 C u le x re stuans 3 To t a l s 0 35 0 0 0 0 R u s s e ll Ae de s albopic t us 9 48 17 19 4 Tox o rhy n c h ite s ru tilus 1 1 To t a l s 0 10 48 17 20 4 S h e l by Ae de s albopic t us 11 42 31 8 C u le x te rrit ans 2 Oc hle r otatus trise r ia tus 5 43 Cule x sali narius 4 Orthopodomy i a s i gnife ra 12 1 3 To t a l s 0 0 32 86 36 8 St . C l a i r Ae de s albopic t us 38 37 38 Orthopodomy i a s i gnife ra 3 To t a l s 0 0 38 37 41 0 S u mte r Ae de s albopic t us 52 38 Oc hle r otatus trise r ia tus 1 C u le x re stuans 9 To t a l s 9 53 0 0 0 0 T a ll ad eg a Ae de s albopic t us 62 C u le x te rrit ans 5 Oc hle r otatus trise r ia tus 20 C u le x re stuans 17 Cule x sali narius 15 Tox o rhy n c h ite s ru tilus 1 To t a l s 37 83 0 0 0 0 T a l l a p oos a Ae de s albopic t us 102 C u le x te rrit ans 20 Tox o rhy n c h ite s ru tilus 2 To t a l s 0 0 124 0 0 0 Tu s c alo o s a Ae de s albopic t us 51 52 57 131 22 C u le x te rrit ans 1 Oc hle r otatus trise r ia tus 2 8 5 C u le x re stuans 34 10 2 Cule x sali narius 8 Orthopodomy i a s i gnife ra 7 C u le x quinque fa sc iatus 1 Tox o rhy n c h ite s ru tilus 1 To t a l s 0 97 70 59 137 29 Wa l k er Ae de s albopic t us 5 C u le x re stuans 53 39 Orthopodomy i a s i gnife ra 29 To t a l s 0 87 0 0 0 0 W a s h i n gt o n Ae de s albopic t us 60 57 51 Oc hle r otatus trise r ia tus 8 Cule x sali narius 1 To tal s 0 61 57 59 0 0 W ilc o x Ae de s albopic t us 43 To tal s 0 0 43 0 0 0 W i ns t o n Ae de s albopic t us 11 C u le x re stuans 23 Cule x sali narius 15 To tal s 0 49 0 0 0 0 40 T a bl e 3. P r es en ce o r ab s e n ce o f t i r e - b r eed i ng m o s q u i t o es by 2- week i n t e r v a l s f r o m M a y t h r o ugh Oct o b e r , b a s e d o n t h e 2004- 2005 l a r v a l s u r v e y . S p ec i e s ar e l i s t e d i n de s c e n d i ng o r d e r o f t h e t o t a l n u m b er s o f eac h s p ec i e s co l l ect ed. M o nt h Mo sq u i t o S p e c i e s Ma y Ju n e Ju l y A u g . S e p t . O c t . A e de s al b o p i c t u s + + + + + + + + + + + C u le x ter r i ta n s + + + + + + + + + + O c h l er o t a t u s tr is er ia tu s + + + + + + + + + Cul e x r e s t uans + + + + + + + + + C u le x s a lin a r iu s + + + + + + + O r t hopodom y i a s i gn i f e r a + + + + + + + C u le x q u i n q u e fa s c ia tu s + + + + + + + O c hl e r ot a t us a t r opal pus + + + + + T o xo r h yn ch i t es r u t ilu s + + + + + + A noph e l e s s p p . + + + + + P s or ophor a c o l u m b i a e + + + A noph e l e s pu nc t i p e nni s + + A noph e l e s qu adr i m a c ul a t us + 41 T a bl e 4. M o s q u i to s p ec i e s co l l ect ed by p h y s i o gr aphi c r e g i o n f r o m t i r e s i n Al a b a m a, b a s e d o n t h e 20 04- 2005 l a r v a l s u r v e y . Spec ie s ar e l i s t e d i n des c e n d i ng o r der o f t h e t o t a l n u m b er s o f eac h s p ec i e s co l l ect ed. P h y s i o gr ap hi c Reg i o n s o f Al a b a m a Mo sq u i t o S p e c i e s Ea s t G u l f C o as t a l P l a i ns A l ab am a V a l l ey a n d R i d g e C u mbe r la nd P l a t e a u H i g h la nd R i m Pi e d m o n t Up l a n d Ae de s albopic t us + + + + + C u le x te rrit ans + + + + + Oc hle r otatus trise r ia tus + + + + + C u le x re stuans + + + + + Cule x sali narius + + + + Orthopodomy i a s i gnife ra + + + + C u le x quinque fa sc iatus + + + + Oc hle r otatus a t ropalpu s + + Tox o rhy n c h ite s ru tilus + + + + + P s orophora c o lumbiae + Anophe le s punc tipe nnis + + + Anophe le s quadrimac ul atus + 42 T a bl e 5. P r es en ce o r ab s e n ce o f t i r e - b r eed i ng m o s q u i t o es c o l l ect ed by p h y s i o gr aphi c r e g i o n by 2- we ek i n t e r v a l s f r o m M a y t h r o ug h Octo b e r , b a s e d o n t h e 2004- 2005 l a r v a l s u r v e y . M o nt h P h y s io g e o g r a p h ic R e g i o n M o s q u ito S p e c i e s M a y J u n e J u ly A u g . S e p t . O c t. E a st Gu l f C o st a l Pl a i n A e de s al b o p i c t u s + + + + + + + + + + + C u le x ter r i ta n s + + + + + + + + + + + O c h l er o t a t u s tr is er ia tu s + + + + + + + + + Cul e x r e s t uans + + + + + + + + + C u le x s a lin a r iu s + + + + + + + + + O r t hopodom y i a s i gn i f e r a + + + + + + + + C u le x q u i n q u e fa s c ia tu s + + + + + + O c hl e r ot a t us a t r opal pus + + T o xo r h yn ch i t es r u t ilu s + + + + + + + P s or ophor a c o l u m b i a e + + A noph e l e s pu nc t i p e nni s + + A noph e l e s s pp + + + + A l ab am a V a lle y an d R i d g e A e de s al b o p i c t u s + + + + + + + + + + C u le x ter r i ta n s + + + + + O c h l er o t a t u s tr is er ia tu s + + + + + Cul e x r e s t uans + + + + + C u le x s a lin a r iu s + + + O r t hopodom y i a s i gn i f e r a + + O c hl e r ot a t us a t r opal pus + + + + + + T o xo r h yn ch i t es r u t ilu s + + A noph e l e s pu nc t i p e nni s + A noph e l e s qu adr i m a c ul a t us + A noph e l e s s pp + C u m b er lan d P l at ea u A e de s al b o p i c t u s + + + + + + + + + + C u le x ter r i ta n s + + + 43 O c h l er o t a t u s tr is er ia tu s + + + + Cul e x r e s t uans + + + + + + C u le x s a lin a r iu s + + + O r t hopodom y i a s i gn i f e r a + + C u le x q u i n q u e fa s c ia tu s + + O c hl e r ot a t us a t r opal pus + H i g h l a n d R im A e de s al b o p i c t u s + + + + + + + + + + C u le x ter r i ta n s + + + + + + + + O c h l er o t a t u s tr is er ia tu s + + + Cul e x r e s t uans + + + C u le x s a lin a r iu s + C u le x q u i n q u e fa s c ia tu s + T o xo r h yn ch i t es r u t ilu s + + + A noph e l e s pu nc t i p e nni s + + P i e d mo n t U p la n d A e de s al b o p i c t u s + + + + + + + + C u le x ter r i ta n s + + + + O c h l er o t a t u s tr is er ia tu s + + + + Cul e x r e s t uans + T o xo r h yn ch i t es r u t ilu s + 44 Figure 1. Map of Alabama Counties reproduced from the Department of Geography, College of Arts and Sciences, the University of Alabama. 45 Figure 2. Map of Alabama physiographic regions reproduced from the Department of Geography, College of Arts and Sciences, the University of Alabama. 46 a b Figure 3. Alabama counties in which larvae were collected from tires, shown in gray. a, 2004. b, 2005. 47 Figure 4. Map of Alabama showing the locations of tire sites sampled in each county during the 2004-2005 larval survey. A total of 169 tire sites was sampled. 48 Aedes albopictus Culex quinquefasciatus Culex restuans Culex salinarius Figure 5. Distribution, by county, of tire-breeding mosquitoes in Alabama, based on 2004-2005 larval survey. Aedes albopictus, Cx. quinquefasciatus, Cx. restuans, and Cx. salinarius. 49 Culex territans Ochlerotatus atropalpus Ochlerotatus Orthopodomyia triseriatus signifera Figure 6. Distribution, by county, of tire-breeding mosquitoes in Alabama, based on 2004-2005 larval survey. Culex territans, Oc. atropalpus, Oc. triseriatus, and Or. signifera. 50 Psorophora columbiae Toxorhynchites rutilus Anopheles punctipennis Anopheles quadrimaculatus Figure 7. Distribution, by county, of tire-breeding mosquitoes in Alabama, based on 2004-2005 larval survey. Psorophora columbiae, Tx. rutilus, An. punctipennis, and An. quadrimaculatus. 51 Anopheles spp. Figure 8. Distribution, by county, of tire-breeding mosquitoes in Alabama, based on 2004-2005 larval survey. Anopheles spp. 52 Figure 9. Occurrence of tire-breeding mosquitoes by 2-week intervals from May through October, based on the 2004-2005 larval survey. Aedes albopictus Culex territans Ochlerotatus triseriatus Culex restuans Culex salinarius Orthopodomyia signifera Culex quinquefasciatus Ochlerotatus atropalpus Toxorhynchites rutilus Psorophora columbiae Anopheles punctipennis Anopheles quadrimaculatus May June July Aug. Sept. Oct. 53 Figure 10. Map of Alabama showing the 5 physiographic regions and the number of tire sites sampled per county, based on the 2004-2005 larval survey. Cumberland Plateau Piedmont Upland Alabama Valley and Ridge East Gulf Coastal Plains Highland Rim 54 EVALUATION OF THE MOSQUITO ATTRACTANT OCTENOL FOR ENHANCING COLLECTIONS OF TIRE-BREEDING MOSQUITOES IN PROPANE-POWERED TRAPS Objectives 1. To determine the species of mosquitoes that are attracted to octenol in an urban setting. 2. To determine if propane-powered traps baited with octenol are an effective tool in monitoring tire-breeding mosquito populations. 3. To determine if propane-powered traps are effective in reducing populations of urban mosquitoes below nuisance levels. 1-octeno-3-ol, (octenol) is a volatile compound that has been isolated from many natural sources, including both invertebrates and vertebrates. Octenol is an 8-carbon mono-unsaturated alcohol with two isomers. Hall et al. (1984) was the first to isolate octenol from oxen breath, and the substance was first successfully used as an attractant in the tsetse control programs in Zimbabwe and other parts of Africa (Torr 1994). Ceratopogonids (Kline 1994), tabanids (French and Kline 1989), oestrids (Anderson 1989), and mosquitoes (Takken and Kline 1989; Kline et. al 1990, 1991) have been shown to be similarly attracted to traps baited with octenol. 55 Field studies conducted in a variety of ecological habitats to evaluate the attractiveness of octenol to mosquitoes, including estuarine ecosystems (Kline et al. 1990, 1991; Takken and Kline 1989, Rueda et al. 2001), freshwater swamps (Takken and Kline 1989), phosphate-mined areas (Kline et al. 1990), and irrigated ricelands (Kline et al. 1991), have shown that many mosquito species are attracted to octenol. Although Kline (1994) found that Ochlerotatus sollicitans, Oc. taeniorhynchus, Oc. triseriatus, Culex salinarius, and Mansonia titillans have shown a positive response to octenol- supplemented traps, other studies showed no effects on collections when octenol alone was used as an attractant (Kline 1994, Kline 2002). In fact, octenol reportedly can cause a negative response in Culex (Kline 1994, Mboera et al. 2000, Burkett et al. 2001). Other studies have shown that the combination of carbon dioxide and octenol results in a synergistic effect in the response of many mosquito species (Kemme et al. 1993, Kline 1994, Kline and Mann 1998). In recent years trapping devices have been developed utilizing catalytic combustion of propane to produce CO 2 , heat, and water vapor as a means of managing some mosquito populations (Kline 2002). The Mosquito Magnet Pro TM (MMP) is one such propane-powered trap that uses a counterflow technology? to emit a plume of CO 2 , heat, and water vapor in combination with octenol. Studies have shown that these octenol- supplemented traps often collect large numbers and a diversity of mosquito species when operated in proximity to salt marshes (Takken and Kline 1989, Kemme et al. 1993, and Rueda et al. 2001). Other studies have shown no differences in collections when octenol is used in other ecological habitats (Burkett et al. 2001, Rueda et al. 2001 and Shone et al 2003). Rueda et al. (2001) found that when trapping in a salt marsh in North Carolina 56 with light traps baited with CO 2 , light and octenol, collections of important vectors of Eastern Equine Encephalitis virus were increased. However, when collecting in a creek flood plain in North Carolina, these authors found no statistically significant difference in mosquito collections with the addition of octenol (Rueda et al. 2001). The MMP traps, produced by the American Biophysics Corporation, are commercially available to homeowners. The pamphlet that comes with the traps cautions ?that recent studies show that Octenol may actually repel the Asian Tiger Mosquito? Aedes albopictus, the number-one nuisance mosquito in many urban communities in the southeastern United States. The source of this statement was unpublished proprietary studies. Only a few studies have addressed the response of Ae. albopictus to octenol- supplemented traps (Shone et al. 2003 and Dennett et al. 2004). Shone (2003) used the Fay-Prince trap baited with CO 2 and CO 2 + octenol to evaluate the ability of the trap to collect Ae. albopictus. They found no statistically significant differences in the response of Ae. albopictus to these two combinations of attractants. Traps using either carbon dioxide alone or CO 2 + octenol were, on the other hand, statistically more attractive to Ae. albopictus than were traps that were either unbaited or used only octenol. These results suggested that CO 2 is driving the response of Ae. albopictus, not octenol. Shone et al. (2003) did not indicate that octenol was acting as a repellent to Ae. albopictus. Thus, there are no published reports that provide a basis for American Biophysics Cooperation?s statement. Propane traps in combination with the attractant octenol have not been extensively evaluated in urban areas. This leaves unanswered questions as to whether or not octenol is attractive or repellent to certain urban mosquitoes. This study was designed to evaluate 57 the effectiveness of the MMP with and without octenol in attracting mosquitoes typically found in urban environments. Three field trials were conducted in the communities of Auburn and Phenix City, Alabama, to evaluate octenol. The specific objectives of the study were to determine: (1) the species of mosquitoes attracted to octenol in an urban setting, (2) if the MMP using octenol as an attractant is an effective tool for monitoring mosquito populations, and (3) if the MMP is an effective trap in reducing mosquito populations of urban mosquitoes below nuisance levels. Study Sites Field Trial 1 Preliminary field trial 1 was conducted using two MMP traps at an auto-repair shop at Auburn, Alabama. Behind of the auto-repair shop was an outdoor tire-storage area where about 200 discarded tires, ranging from compact-car tires to tractor-trailer tires, were stored. About half of these tires were sheltered in a covered tire rack. Because the tires were protected, the tires did not hold water and subsequently were not sites of mosquito larval development. The remaining tires were stored in an open area adjacent to the tire rack. These tires were either laying flat on the ground or propped up against other tires. Mosquito larvae were observed in many of these tires. A fence covered with kudzu (Pueraria montana) bordered the perimeter of the auto shop just behind the area where the tires were located. 58 Field Trial 2 Preliminary field trial 2 was conducted using two MMP traps placed in the proximity of 4 greenhouses located on the Auburn University Campus. Two MMP traps were placed 2 meters apart in a low-lying drainage area that collected runoff from the irrigation system used in the greenhouses. Small runoff pools were formed in this area allowing mosquito breeding. Loblolly pine (Pinu taeda), American holly (Ilex opaca), pin oak (Quercus palustris), and willow oak (Quercus phellos) were the predominant vegetation in the drainage area. Ornamental ponds that held about 4-6 inches of standing water were located near the greenhouses and were possible mosquito breeding sites. Field Trial 3 The salvage yard is a 3-acre fenced lot with cars, car parts, and discarded tires scattered throughout. The site mostly consisted of wrecked cars that were lined up in rows in the front and back of the lot. At the front end of the lot approximately 25 tires were stacked horizontally on top of each other and held very little rain water. Along the far back fence corner there were approximately 40 tires of varying sizes that consistently held water during the 8-week field trial. Mosquito larvae were observed in these tires. In the center of the salvage yard a wooded area consisting of predominantly sweet gum (Liquidamtar styraciflua) and swamp willow (Salix caroliniana) separated the front lot from the back lot. Other vegetation in this area included tulip tree (Liriodendron tulipifera), goldenrod (Solidago spp.), cattails (Typha latifolia), sedge (Carex firma), and rush (Juncus patens). Approximately 500 tires were scattered throughout the adjacent wooded area. These tires were lying on their sides, propped up against one another, or 59 piled haphazardly on top of one another in tire mounds. Mosquito larvae also were observed in these tires. Materials and Methods Field trials were conducted during the summer of 2004 to evaluate the performance of the propane-powered Mosquito Magnet Pro TM trap (Figure 11) (American Biophysics Corporations, East Greenwich, RI). The catalytic combustion of propane, which converts 20 pounds of propane to 60 pounds of CO 2, generates the power to run the counterflow suction fan for insect entrapment while producing the long-range attractants. Replaceable 1.7-g octenol cartridges (American Biophysics Corporation) were placed in the compartment located at the bottom of the MMP fan unit. The first two preliminary trials were conducted at two locations in Auburn, AL. Two MMP traps were placed at each trial site. One trap was operated with octenol, whereas the other trap was not. Collection nets were removed and replaced each day. All mosquito collections were brought to the laboratory for identification to species. Each of these two trials was conducted over a 4-week period, the first trial from May 27 to June 23 and the second trial from June 2 to June 23. The third field trial was conducted at an automobile salvage and tireyard at Phenix City, AL from July 7 to August 16. Four MMP traps were placed 20 meters apart in a 1x2 factorial design in the wooded area located in the center of the salvage yard. Four traps were operated weekly, two traps with octenol and two traps without octenol, throughout the 8-week field trial. Each trap was supplemented with octenol, such that 60 octenol occupied each trapping position at least one time during the field trial. Octenol was replaced at the end of each 7-day period. Mosquito collections were removed at the end of each 7-day period, and the species identified and counted. Two two-minute landing counts (Figure 12) were taken twice a week between 9:00 and 11:00 am within the salvage yard, with locations randomly selected on each occasion by tossing a stick. The right or left leg from knee down was exposed while a hand-held battery aspirator was used to remove the landing mosquitoes. A two-minute acclimation time was allowed before the landing counts began. The landing counts were averaged by week. Six tires were randomly sampled each week. All the water was removed from the tires and the mosquito larvae recovered. The samples were brought back to the lab for larval identification. Tires were sampled throughout the trial to determine which mosquito species were breeding in the tires at the salvage yard. Statistical Analysis For preliminary field trials 1 and 2 collections with greater than 100 mosquitoes, a repeated measures ANOVA was used to determine if there was a significant difference between the treatments. The repeated measures ANOVA was used because the dependent variable, time, was repeated. For the third field trial collections with greater than 100 mosquitoes treatment (octenol vs. no octenol), position, and week effects were analyzed using a 3-way ANOVA for each species trapped. The 3-way ANOVA was used to determine the effectiveness of octenol in enhancing collections of individual mosquito species. A Tukey?s test was used if there were any significant interactions 61 between the variables tested in the 3-way ANOVA. A Chi-square test was used for species with trap collections less than 100. A Proc Corr analysis was used to determine if there was any correlation between the mean weekly landing counts for each mosquito species and the collections in the MMP traps. Results Preliminary Field Trial 1 A total of 1501 mosquitoes, representing 7 species in 5 genera, was collected during the 4-week field trial (Table 6). A total of 1061 (71%) of the mosquitoes was collected in MMP traps provided with octenol, compared to 440 (29%) collected in the MMP traps without octenol. The most frequently collected species in the octenol- supplemented traps was Ae. albopictus (1472/1501), comprising 98% of the collections. Collections of Ae. albopictus were 2.5-fold greater with octenol (1051, or 71%) than without octenol (421, or 29%). On 5 individual trap nights, collections of Ae. albopictus were significantly enhanced with use of octenol (Figure 13). The next most frequently collected species were Anopheles punctipennis, Cx. quinquefasciatus, Ae. vexans, Psorophora columbiae, and Ochlerotatus triseriatus. There was a significant difference between treatments for collections of Ae. albopictus [P=.03] as determined by a repeated measures ANOVA (Table 7). There was no significant difference between the treatments of Cx. quinquefasciatus, Ae. vexans, Ps. columbiae, and Oc. triseriatus. Anopheles punctipennis collections were significantly decreased when octenol was used as the attractant, [P =.01]. 62 Preliminary Field Trial 2 A total of 655 mosquitoes, representing 6 species in 5 genera, was collected during the 3-week field trial (Table 8). A total of 552 (84%) of the mosquitoes was collected using octenol. Only 103 (16%) mosquitoes were collected without octenol. Of these collections 570 (80%) were Ae. albopictus, with 489 (86%) collected with octenol, versus 81 (14%) collected without octenol. On 9 individual trap-nights, Ae. albopictus collections were significantly enhanced with octenol (Figure 14). The next most frequently collected species were: An. punctipennis (35), Cx. quinquefasciatus (27) Ae. vexans (19), Oc. triseriatus (2), and Ps. columbiae (2). There was a significant difference between treatments (octenol vs. no octenol) on the collections of Ae. albopictus [P =.02] as determined by a repeated measures ANOVA (Table 9). Octenol significantly increased the collections of Ae. albopictus by 6 fold. The treatments were not significant in the collections of Cx. quinquefasciatus, Oc. triseriatus, and Ps. columbiae. However, the addition of octenol significantly increased the collections of An. punctipennis [P=.05] by 3 fold and Ae. vexans [P=.05] by 18 fold. Field Trial 3 A total of 7143 mosquitoes, representing 13 species in 5 genera, was collected over the 8-week study period (Table 10). A total of 5773 mosquitoes (81%) was collected with octenol, versus 1370 (19%) in the MMP traps operated without octenol. Of these collections 5571 (78%) were Ae. albopictus, with 4334 (77%) collected with octenol. The next most frequently collected species were, in descending order: Oc. triseriatus 63 (1302), Coquillettida perturbans (131), An. punctipennis (35), Ps. columbiae (33), Cx. restuans (29), Cx. salinarius (13), Cx. erraticus (9), Cx. quinquefasciatus (8), An. crucians (5), Ae. vexans (3), Ps. ferox (3), and Cx. territans (1). Culex salinarius and Cx. territans were collected only with MMP traps provided with octenol. All 13 mosquito species trapped, with the exception of Culex quinquefasciatus, were collected in greater numbers with octenol than without octenol. There were 4 times more mosquitoes collected with octenol than without octenol. There were no statistically significant effects between treatment (octenol vs. no octenol) and trap position as detected by the 3-way ANOVA for Ae. albopictus, Oc. triseriatus, and Cq. perturbans (Table 11). However, there were significant effects between treatment and week indicated by the 3-way ANOVA for Ae. albopictus and Oc. triseriatus. Tukey?s test detected a significant difference between week 1 and all other weeks. There were no effects between treatment and week for Cq. perturbans. There was an effect between treatments (octenol vs. no octenol) for collections of Ae. albopictus, Oc. triseriatus, and Cq. perturbans based on a 3-way ANOVA. Significantly more Ae. albopictus were collected with octenol [P<.01]. This was also true for collections of Oc. triseriatus [P<.05] and Cq. perturbans [P<.01]. Aedes albopictus collections were increased 3.5 fold with octenol. Ochlerotatus triseriatus collections were increased 12 fold with use of octenol. Octenol increased Cq. perturbans collections by 20 fold. Based on a Chi-square analysis of mosquito species trapped in low number , i.e., totals less than 100 specimens of An. punctipennis, Cx. restuans , Cx. salinarius, and Ps. columbiae were all significantly enhanced by use of octenol [P=.05]. 64 The Proc Corr analysis comparing week, collections, and landing counts showed a significant correlation between collections and landing counts of Ae. albopictus [P<.01], (Figure 15). Since only Ae. albopictus was collected during the landing counts, no comparisons could be made between collections and week for other species trapped throughout the 8-week trial. Mean landing counts/ 2 minutes ranged from a high of 22 Ae. albopictus to a low of 4 Ae. albopictus. The overall landing-count average during the 8-week study was approximately 12 Ae. albopictus biting every two minutes. A total of 1466 mosquito larvae, representing 7 species in 6 genera, was collected from tires at the salvage yard during the study period. They were, in decreasing order, Ae. albopictus (902, Cx. territans (289), Oc. triseriatus (211), Or. signifera (46), Cx. restuans (13), Tx. rutilus (3), and Anopheles species (2) (Table 12). Discussion Previous studies have shown that the combination of CO 2 and octenol significantly increased collections of Cq. perturbans, An. punctipennis, Ps. columbiae, Cx. restuans, and Cx. salinarius (Kline et al. 1990, Kline et al. 1991, Kline 1994, Rueda et al 2001). The results of the field trials in our study support these reports. The use of octenol with the MMP trap was effective in enhancing the response of many mosquito species, making it an effective trap for general monitoring of mosquito populations. The results of preliminary field trials 1 and 2 indicated that there was a general trend of increased response of most species collected with the MMP and octenol, in that 75% of mosquito collections throughout the trials were trapped with octenol. Octenol 65 significantly enhanced collections of Ae. albopictus at Field Site 1 and 2. Aedes vexans, An. punctipennis, and Cx. quinquefasciatus collections were significantly enhanced at Field Site 2. Collections of these species at Field Site 1 were not statistically different. However, with the exception of An. punctipennis, these species were collected in greater numbers with octenol-baited traps. There was a negative effect of octenol observed in the collections of An. punctipennis in Field trail 1. Kline et al. (1991) found that octenol at times enhanced collections of anopheline mosquitoes and at other times appeared to repel these species. This observation is supported by the data reported here. The mosquitoes trapped at Field Site 3 showed a positive response to the MMP baited with octenol, such that 81% of mosquito collections in this study were collected with octenol. Aedes albopictus, Oc. triseriatus, Cq. perturbans, An. punctipennis, Ps. columbiae, Cx. restuans, and Cx. salinarius showed a positive significant response to octenol-baited traps with overall collections increased 4 fold. To our knowledge, this is the first published report of Ae. albopictus and Oc. triseriatus being significantly attracted to combinations of CO 2 and octenol. Since Ae. albopictus was trapped more during all 3 field trials with octenol, the previous claim that octenol is repellant to Ae. albopictus is not supported in this study. In fact in field trial 3, Ae. albopictus was trapped more with octenol throughout each week?s trap rotation with a 3-fold increase in collection numbers throughout the study. The claim that Ae. albopictus is repelled by octenol may be attributed to the fact that most studies evaluating the Mosquito Magnet? traps baited with octenol were conducted in salt marshes where Ae. albopictus is not commonly collected. The studies reported here show that in an urban 66 environment octenol does enhance collections of Ae. albopictus and is driving the response of Ae. albopictus collections. Propane-powered traps did not reduce the natural populations of Ae. albopictus below the nuisance levels at field site 3. If the MMP reduced the natural populations of Ae. albopictus below nuisance levels a larger number of Ae. albopictus would have been collected in the MMP traps. Likewise a reduction in the numbers of Ae. albopictus landing during the landing counts would have been evident. This was not observed during field trial 3. There was a significant correlation between landing counts and MMP collections of Ae. albopictus. Even though the collections of Ae. albopictus in the MMP were high, Ae. albopictus biting activity was still considered to be above the nuisance level. Because our field sites were not typical of what homeowners face, it cannot be concluded that these traps would not reduce nuisance populations in a residential setting. If people live in an area where one or more mosquito species exhibit high numbers, the chances of reducing those populations to personally acceptable is low. In fact, Dennett et al.?s (2004) daily observations suggested that counterflow traps were efficient in not only capturing mosquitoes but also attracting mosquitoes that were never captured. Based on his observations, it is possible that the MMP trap attracts more mosquitoes than the trap actually captures, increasing overall mosquito abundance in the vicinity of the traps(s). The results of this study indicated that octenol-baited MMP traps enhanced mosquito collections in all field trials. Because octenol causes a positive response in many species, the MMP trap can be an effective tool in monitoring mosquito populations. However, these traps alone may not be adequate for urban homeowners to control nuisance mosquitoes. 67 Literature Cited Anderson JR. 1989. Use of deer models to study larviposition by wild nasopharyngeal bot flies (Diptera: Oestridae). J Med Entomol 26:234-236. Burkett DA, Lee WJ, Lee KW, Kim HC, Lee HI, Lee JS, Shin EH, Wirtz RA, Cho HW, Claborn DM, Coleman RE, Klein TA. 2001. Light, carbon dioxide, and octenol-baited mosquito trap and host-seeking activity evaluations for mosquitoes in a malarious area of the republic of Korea. J Am Mosq Control Assoc 17:196-205. Dennett JA, Vessey NY, Parsons RE. 2004. A comparison of seven traps used for collection of Aedes albopictus and Aedes aegypti originating from a large tire repository in Harris County (Houston), Texas. J Am Mosq Control Assoc 20:342-349. French FE, Kline DL. 1989. 1-octen-3-ol, an effective attractant for Tabanidae (Diptera). J Med Entomol 26:459-461. Gillies, MT. 1980. The role of carbon dioxide in host-finding by mosquitoes (Diptera: Culicidae): a review. Bull Entomol Res 70:525-532. Hall DR, Beevor PS, Cork A, Nesbitt BF, Vale GA. 1984. 1-octen-3-ol: a potent olfactory stimulant and attractant for tsetse isolated from cattle odors. Insect Sci Appl 5:335-339. Kemme JA, Van Essen PHA, Ritchie SA, Kayo BH. 1993. Response of mosquitoes to carbon dioxide and 1-octen-3-ol in southeast Queensland, Australia. J Am Mosq Control Assoc 9:431-435. Kline DL. 1994. Olfactory attractants for mosquito surveillance and control: 1-octen-3- ol. J Am Mosq Control Assoc 10:280-287. Kline DL. 2002. Evaluation of various models of propane-powered mosquito traps. J Vect Ecol 1-7. 68 Kline DL, Dame DA, Meisch MV. 1991. Evaluation of 1-octeno-3-ol and carbon dioxide as attractants for mosquitoes associated with irrigated rice fields in Arkansas. J Am Mosq Control Assoc 7: 165-169. Kline DL, Mann MO. 1998. Evaluation of butanone, carbons dioxide and 1-octen-3-ol as attractants for mosquitoes associated with North Central Florida Bay and cypress swamps. J Am Mosq Control Assoc 14:289-297. Kline DL, Takken W, Wood JR, Carlson DA. 1990. Field Studies on the potential of butanone, carbon dioxide, honey extract, 1-octen-3-ol, lactic acid, and phenols as attractants for mosquitoes. Med Vet Entomol 4:383-391. Mboera LE, Takken W, Sambu EZ. 2000. The response of Culex quinquefasciatus (Diptera: Culicidae) to traps baited with carbon dioxide, 1-octen-3-ol, acetone, butyric acid, and human foot odour in Tanzania. Bull Entomol Res 90:155-159. Rueda LM, Harrison BA, Brown JS, Whitt PB, Harrison RL, Gardner RC. 2001. Evaluation of 1-octen-3-ol, carbon dioxide, and light as attractants for mosquitoes associated with two distinct habitats in North Carolina. J Am Mosq Control Assoc 17:61-66. Shone SM, Ferrao PN, Lesser CR, Glass GE, Norris DE. 2003. Evaluation of carbon dioxide and 1-octen-3-ol-baited Centers for Disease Control Fay-Prince traps to collect Aedes albopictus. J Am Mosq Control Assoc 19:445-447. Takken W, Kline DL. 1989. Carbon dioxide and 1-octen-3-ol as mosquito attractants. J Am Mosq Control Assoc 5:311-316. Torr SJ. 1994. The tsetse (Diptera: Glossinidae) story: implications for mosquitoes. J Am Mosq Control Assoc 10:258-265. 69 Table 6: Mosquito species collected with and without octenol at Field Site 1, June 2004. Species are listed in descending order of the total number of each species collected. With Octenol Without Octenol Mosquito Species No. (%) No. (%) Aedes albopictus 1051 (71) 421 (29) Anopheles punctipennis 0 (0) 14 (100) Culex quinquefasciatus 4 (57) 3 (43) Aedes vexans 2 (50) 2 (50) Culex restuans 2 (100) 0 (0) Psorophora columbiae 1 (100) 0 (0) Ochlerotatus triseriatus 1 (100) 0 (0) Totals 1061 440 70 Table 7. Repeated measures ANOVA values for mosquito species with trap collections >100 specimens at Field Site 1. Treatment Treatment x Time Mosquito Species P F DF P F DF Aedes albopictus 0.0321 29.64 1 0.5384 0.89 7 71 Table 8. Mosquito species collected with and without octenol at Field Site 2, June-July 2004. Species are listed in descending order of the total number of each species collected. With Octenol Without Octenol Mosquito Species No. (%) No. (%) Aedes albopictus 489 (86) 81 (14) Anopheles punctipennis 27 (77) 8 (23) Culex quinquefasciatus 17 (63) 10 (37) Aedes vexans 18 (95) 1 (5) Ochlerotatus triseriatus 0 (0) 2 (100) Psorophora columbiae 1 (50) 1 (50) Totals 552 103 72 Table 9. Repeated measures ANOVA values for mosquito species with trap collections >100 specimens at Field Site 2. Treatment Treatment x Time Mosquito Species P F DF P F DF Aedes albopictus 0.0280 34.24 1 0.0872 2.67 5 73 Table 10. Mosquito species collected with and without octenol at Field Site 3, July- August 2004. Species are listed in descending order of the total number of each species collected. With Octenol Without Octenol Mosquito Species No. (%) No. (%) Aedes albopictus 4334 (78) 1237 (22) Ochlerotatus triseriatus 1202 (92) 100 (8) Coquillettida perturbans 125 (95) 6 (5) Anopheles punctipennis 33 (94) 2 (6) Psorophora columbiae 28 (85) 5 (15) Culex restuans 20 (69) 9 (31) Culex salinarius 13 (100) 0 (0) Culex erraticus 8 (89) 1 (11) Culex quinquefasciatus 1 (12) 7 (88) Anopheles crucians 4 (80) 1 (20) Aedes vexans 2 (67) 1 (33) Psorophora ferox 2 (67) 1 (33) Culex territans 1 (100) 0 (0) Totals 5773 1370 74 Table 11. ANOVA values for mosquito species with trap collections > 100 specimens at Field Site 3. Treatment Treatment x Pos Treatment x Week Species P F DF P F DF P F DF Ae. albopictus 0.0003 38.04 1 0.402 1.11 3 0.0114 6.1 6 Oc. triseriatus 0.0003 38.56 1 0.769 0.38 3 0.0004 16.13 6 Cq. perturbans < 0.0001 51.06 1 0.4318 1.02 3 0.0541 3.47 6 75 Table 12. Mosquito larval samples collected from tires at parts-and-salvage yard in Phenix City, Alabama, during Field Trial 3 evaluating the Mosquito Magnet Pro TM traps with and without octenol. Species No. Aedes albopictus 902 Culex territans 289 Ochlerotatus triseriatus 211 Orthopodomyia signifera 46 Culex restuans 13 Anopheles spp 2 Toxorhynchites rutilus 3 Totals 1466 76 Figure 11. American Biophysics Corporation?s propane-powered Mosquito Magnet Pro TM trap. 77 Figure 12. Collections of landing mosquitoes using a hand-held, battery-operated aspirator to remove landing females during a 2-minute period to determine biting activity at a parts-and-salvage yard, in Phenix City, Alabama, 2004. 78 0 50 100 150 200 250 300 350 400 450 5/27 5/31 6/4 6/8 6/12 6/16 6/20 6/24 Successive Trap Night N u m b er o f A e . al b o p i ct u s t r ap p e d With Octenol Without Octenol * * ** * Figure 13. Collections of Aedes albopictus with and without octenol at Field Site 2 during successive trap nights. Collections of Aedes albopictus (black) were made with octenol. Collections of Ae. albopictus (gray) were made without octenol. Five trap-night collections were significantly enhanced with octenol (P < .05), as indicated by asterisks, based on a Chi-square analysis. 79 Figure 14. Collections of Aedes albopictus with and without octenol at Field Site 2 during successive trap nights. Collections of Aedes albopictus (black) were made with octenol. Collections of Ae. albopictus (gray) were made without octenol. Nine trap-night collections were significantly enhanced with octenol (P < .05), as indicated by asterisks, based on a Chi-square analysis. 0 20 40 60 80 100 120 6/2 6/7 6/12 6/17 6/22 6/27 Successive Trap Night Num b e r of Ae des a l bopi ct us t r ap ped With Octenol Without Octenol * * * * * * * * * 80 0 500 1000 1500 2000 2500 3000 1234567 Week M M P Col l e c t i ons ( N o. m o s qui t o e s ) 0 5 10 15 20 25 La nd i n g Cou n t ( A v g . p e r / 2 m i ns ) Figure 15. MMP collections compared to mean landing counts of Ae. albopictus by trap week. MMP collections on the left y-axis and mean landing-count collections on right y- axis of Aedes albopictus at Field Site 3. Based on a Proc Corr analysis, the mean landing counts of Ae. albopictus were significantly correlated with the MMP collections by week, P < .05. Landing-count averages of Ae. albopictus 81 APPENDIX List of mosquito species mentioned in the text and their author (Darsie and Ward 1981). Mosquito species are listed in alphabetical order. Mosquito Species Author Aedes albopictus (Skuse) Aedes aegypti (Linnaeus) Aedes vexans (Meigen) Anopheles barberi Coquillett Anopheles crucians Wiedemann Anopheles punctipennis (Say) Anopheles quadrimaculatus Say Culex erraticus (Dyar and Knab) Culex pipiens Linnaeus Culex restuans Theobald Culex quinquefasciatus Say Culex salinarius Coquillett Culex territans Walker Coquillettida perturbans (Walker) Culiseta melanura (Coquillett) Ochlerotatus atropalpus (Coquillett) Ochlerotatus bahamensis Berlin Ochlerotatus japonicus (Theobald) Ochlerotatus sollicitans (Walker) Ochlerotatus taeniorhynchus (Wiedmann) Ochlerotatus triseriatus (Say) Orthopodomyia signifera (Coquillett) Mansonia titillans (Walker) Psorophora columbiae (Dyar and Knab) Psorophora ferox (von Humboldt) Toxorhynchites rutilus (Coquillett)